Three-photon head-mounted microscope for imaging deep cortical layers in freely moving rats

a, average image of pollen grains acquired from the miniature microscope using a single-ring hollow-core photonic crystal fiber with a 25 µm core diameter, showing the region of interest (ROI, green) in which the fluorescence trace in b was quantified. b, fluorescence trace before and during (gray box) bending of the fiber approximating that experienced during freely moving experiments. Insert shows a micrograph of a cross section through the fiber. c, pollen grains imaged using a HC-PBGF with a 14 µm core-diameter. ROI in which the fluorescence trace in d shown in green. d, fluorescence trace before and during (gray box) bending of the fiber. Insert shows a micrograph of a cross section through the fiber. e, spectral phase of the pulses propagating through 1.1 m of fiber, with compensation of group velocity dispersion (GVD) only (green), third-order dispersion (TOD) and GVD compensated (blue) and the flat phase corresponding to the transform-limited pulse (red) in Fig. 1d. The insert shows the same curves with magnified Y-axis scaling and the normalized spectrum used for the measurements of the spectral phases (black). f, transmission losses of the fiber as a function of wavelength. Plot shows mean (dark blue) and measurement uncertainty, calculated as described previously¹³. g, full laser pulse temporal profile after the fiber before dispersion compensation (green) and after compensation (blue) compared to calculated transform-limited pulse with the spectrum measured after the fiber (red). h, example laser spectra recorded after transmission through the HC-PBGF for incident power giving 0.5 (blue), 15.7 (red) and 87.5 mW (yellow) transmitted power at the sample. i, laser spectrum center of mass wavelength recorded after transmission through the HC-PBGF as a function of incident power (power measured at the sample). Panels a-d representative of 2 separate experiments.

a, optical design for miniature microscope with parts listing.

a, log-log plot of gray scale value (GSV) of fluorescence intensity as a function of laser power demonstrating 3-photon excitation. Least squares fit has a slope of 2.99. b, probability distribution of photon counts after the laser pulse trigger. Shaded region shows time window for laser pulse triggered data acquisition. Insert shows raw data from 2609 individual laser pulses. c, example images of the same pollen grain acquired with standard (left) and gated (right) acquisition. e, box and whisker plot of image signal-to-noise ratio for gated and standard image acquisition. Red line indicates data median, box shows 25^th and 75^th percentile of data and whiskers represent an extension to 1.5 times the interquartile range (outliers are plotted as red markers). f–h, overview images showing dendrites (arrowheads) visible at the indicated depths from the cortical surface. Scale bar in g 20 µm, applies to all panels. Panels f–h are representative of >6 datasets from 6 animals.

a, overlay of movement paths for all datasets from all animals, each path represented in a different color (n=7 datasets from 5 animals). Track outline shown in black. Paths can leave the track boundary when the animal reaches its head over the track edge to peer around. b, distributions showing animals’ velocity (left) and acceleration (right) during the imaging files for which the behavioral paths are shown in a. Distributions for individual animals shown in gray, mean in black. c, distributions of head roll (left), pitch (middle) and azimuth (right) over all datasets shown in a, during periods when the animals’ velocity was greater than 0.05 ms^-1. Positive roll is roll to the animal’s right, positive pitch is up and azimuth 0° is up in a. Distributions for individual animals shown in cyan, mean in black. d, same as for c, but including all data, including extended periods where the animal was grooming or peering in one direction off the track. e, distribution of image displacement (mean ± SD) for different animal velocities for all datasets (7 datasets from 5 animals). Colors denote different datasets. f, mean image displacement (mean ± SD) as a function of animal acceleration. Datasets and colors as in e.

a, raw fluorescence trace (black) and low-pass filtered fluorescence signal (Gaussian filter, 150 ms time constant, red). Blue crosses show the negative transients selected by the following analysis. b, high-pass filtered trace obtained by subtracting a low-pass filtered fluorescence from the raw trace. c, high-pass filtered signal from b scaled with the square root of the low-pass filtered fluorescence signal and the chosen threshold (red line). d, fluorescence trace obtained by low-pass filtering the raw signal (Gaussian filter, 30 ms). Black crosses show timings, when events were selected. e, fluorescence trace obtained by applying a rolling-average of 2 frames on the raw data. Black crosses show timings, when events were selected. f, individual examples (blue) and mean (red) of relative changes of fluorescence centered around the selected events from one neuron over 200 s of imaging data (<20 events from 6000 frames). Mean from rolling 2 frame average filtered data for the same data segments shown in black. g, average event trace (red) and the corresponding filtered traces with a rolling average of 2 frames filter (black), 30 ms Gaussian filter (green), 50 ms Gaussian filter (cyan) and 75 ms Gaussian filter (magenta). h, sequence of 7 consecutive images from the frame sequence averaged around detected events. Frame 4 corresponds to the average detected event. Neuron of interest outlined in red in frame 4. i, heat map of the probability of occurrence of events over the track. j, histograms of probabilities of the occurrence of events over the range measured during imaging for acceleration (upper left), velocity (lower left), angular acceleration (upper right) and angular velocity (lower right).

a, individual diagrams of headplate and associated microscope mounting plates. i, implanted titanium headplate. ii, intermediate plate. iii, objective mount plate. iv, objective thread. v, miniature objective with threaded brass sleeve. vi, removable 30 × 5 mm² tab for anesthetized experiments (alternative 15 × 5 mm² tab used for awake experiments). b, cross section through headplate and microscope mounting plates as assembled during imaging experiments.

a, detector system design and parts listing. b, example multi-channel green, red and blue data acquisition using the miniature microscope, with merged image overlay. Green labelling is GCaMP6s labelled neurons, red from SR101 labelled astrocytes and blue from third-harmonic signal, all in rat posterior parietal cortex. c, dependence of red fluorescence emission as a function of excitation laser power at 1320 nm. Data in panel b representative of 4 experiments in 4 animals. Data in panel c from a single measurement.